This invention relates to synthetic indolocarbazole analogues and uses thereof. More particularly, this invention relates to modifications of the core structure 12-(xcex2-D-gluocopyranosyl)-6,7,12,13-tetrahydroindolo[2,3-xcex1]pyrrolo[3,4-c]carbazole-5,7-dione, containing substitutions consisting of a 2,3,9-trihydroxy pattern, particularly cyclic and acyclic ethers at the 2- and 3-hydroxy positions. This invention also relates to compositions and methods of using such indolocarbazole analogues for the inhibition of topoisomerase I activity.
Human topoisomerase I (Topo I) is an enzyme critical to the viability of cellular function that is an attractive target for the design and development of anticancer therapeutics. Currently there are two anticancer agents approved by the Food and Drug Administration for the clinical treatment of cancers: topotecan (Hycamtin) and CPT-11 (Camptosar), both of which are structural analogues of the natural product camptothecin.
Topo I is a 100 kD monomeric protein that catalyzes changes in the topological state of double-stranded DNA (dsDNA) in increments of one linking number1. The three-dimensional structure of human Topo I has been reported2. The mechanism by which Topo I acts is believed to proceed through induction of a transient single-stranded break in dsDNA via formation of a covalent protein-DNA adduct referred to as the cleavable complex, so named because these complexes are detected as DNA breaks upon treatment with denaturing agents or alkali. The cleavable complex is formed upon transesterification of a DNA phosphodiester linkage by the active site tyrosine-723 residue on human Topo 1, resulting in an ester linkage between the enzyme and the 3xe2x80x2-phosphoryl end of the broken DNA strand. This allows free rotation of the protein-bound 3xe2x80x2 end of the broken DNA strand about the intact complementary DNA strand, resulting in relaxation of the duplex in increments of one linking number. Religation of the broken strand (via a second transesterification reaction) and subsequent dissociation of topoisomerase I completes the catalytic cycle.
Topoisomerase I poisons act via stabilization of the cleavable complex, mediated by formation of a ternary complex consisting of drug, topoisomerase I and DNA3. Agents such as camptothecin (the prototype topoisomerase I poison) do not bind to DNA directly, nor to topoisomerase I alone, but only to topoisomerase I complexed with DNA. It has been postulated that the stabilized DNA-protein-drug complex causes lethal DNA strand breaks upon collision with the advancing replication fork. It is by this mechanism that the topoisomerase I poison converts the enzyme into a DNA damaging agent, resulting in disruption of DNA replication and, eventually, cell death. This postulate is supported by the fact that camptothecin is highly phase-specific, only killing cells in S-phase.
In addition to the camptothecins, indolocarbazoles have also demonstrated potent antitumor activity via the poisoning of topoisomerase I activity4-7, most notably ED-1108NB-5069, and J-10708810. The indolocarbazole analogue bearing a 3,9-dihydroxy substitution pattern was found to have superior topoisomerase I poisoning capability as well as superior in vitro antitumor activity relative to the other xe2x80x9csymmetricalxe2x80x9d dihydroxylated regioisomers11. The 3,9-dihydroxy analogue also exhibited impressive in vivo antitumor activity against the DU-145 human prostate tumor line xenotransplanted into nude mice.
The present invention relates to compounds, compositions and methods for the inhibition of topoisomerase I activity.
Accordingly, one object of the invention is to provide compounds of the general formulas I and II, 
wherein R1 and R2 are independently H, C1-6 alkyl, aryl-C1-6 alkyl, mono- or polyfluorinated C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, cyclo(C3-6)alkyl,
or an aryl, wherein the aryl comprises of six membered aromatic carbocycle such as phenyl or a polycyclic aromatic hydrocarbon such as naphthyl, phenanthracenyl, indanyl
or a heterocycle wherein the heterocyle comprises of six membered aromatic heterocycles such as piridyl, diazinyl, pyrimidinyl, pyrrolidinyl, piperazinyl, or five membered aromatic heterocyles such as pyrrolyl, pyrazole, imidazolyl, imidazolidinyl, imidazolenyl, oxazolyl, isoxazolyl, thiazolyl, thiazolidinyl, thiazolinyl, isothiazolyl, isothiazolidinyl, isothiazolinyl, furanyl, thiophenyl or bycyclic systems such as indolyl, benzthiopheneyl, benzofuranyl, isoindolyl, isobenzothiophenyl, isobenzofuranyl; wherein the aryl or the heterocycle may each be unsubstituted or substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl;
or R1 and R2 combine to form a ring ranging in total ring size from five (R1xe2x95x90R2xe2x95x90CH2) to nine (R1xe2x95x90R2xe2x95x90(CH2)5), wherein one or more of the methylene(CH2)hydrogen atoms may be replaced with halogen, C1-6 alkyl, aryl-C1-6 alkyl, mono- or polyfluorinated C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, cyclo(C3-6)alkyl,
or an aryl, wherein the aryl comprises of any six membered aromatic carbocycle such as phenyl or a polycyclic aromatic hydrocarbon such as naphthyl, phenanthracenyl, indanyl
or a heterocycle wherein the heterocyle comprises of six membered aromatic heterocycles such as piridyl, diazinyl, pyrimidinyl, pyrrolidinyl, piperazinyl, or five membered aromatic heterocyles such as pyrrolyl, pyrazole, imidazolyl, imidazolidinyl, imidazolenyl, oxazolyl, isoxazolyl, thiazolyl, thiazolidinyl, thiazolinyl, isothiazolyl, isothiazolidinyl, isothiazolinyl, furanyl, thiophenyl or bycyclic systems such as indolyl, benzthiopheneyl, benzofuranyl, isoindolyl, isobenzothiophenyl, isobenzofuranyl; wherein the aryl or the heterocycle may each be unsubstituted or substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl;
R3 comprises H
or NH2, nitrilo (C1-6)alkyl amine of the formula xe2x80x94NH-alkyl-CN, 2-pyrrolidinyl ethyl-1-amine, benzyl amine, 2-naphthyl amine, 2-benzothiazole amine, beta phenethyl amine, 1-piperazine amine, 4-(2-hydroxyethyl)piperazine-1-amine, piperidine-1-amine, aniline, 2-hydroxy butyl amine, 3-sulfolane amine, 4-methyl 2,3 dihydro isocytosine, (C1-6)alkyl sulfonamidyl, 2-(1H-1,2,4-triazol-1yl)acetamidyl, 1,4-dimethylpiperazine-2-formamidyl, phenoxyformamidyl, gluconamidyl, manonamidyl, gulonamidyl,
or aryl carbamate: of the formula xe2x80x94NHxe2x80x94COxe2x80x94Ar, wherein the aryl comprises of six membered aromatic carbocycles comprising phenyl, hydroxy phenyl, dihydroxy phenyl, trihydroxy phenyl, or a polycyclic aromatic hydrocarbon such as naphthyl, phenanthracenyl, indanyl,
or heterocycle carbamate of the formula xe2x80x94NHxe2x80x94CO-hetrocycle: wherein the heterocyle comprises of six membered aromatic heterocycles comprising piridyl, diazinyl, pyrimidinyl, 5-methoxy pyrimidinyl, pyrrolidinyl, (1,2,4)triazine-3,5-dione-6-yl, 6-mercaptopyrimidine-4yl, or six membered non-aromatic heterocycles comprising piperazinyl, 4-methyl piperazinyl, pyranyl, or five membered aromatic heterocyles comprising pyrrolyl, 1-methyl pyrrol-2-yl, pyrazolyl, imidazolyl, imidazolidinyl, imidazolenyl, oxazolyl, isoxazolyl, thiazolyl, 2-methyl thiazol-4-yl, thiazolidinyl, thiazolinyl, isothiazolyl, isothiazolidinyl, isothiazolinyl, furan-2-yl, thiophen-2-yl, thiophen-3-yl, 3xe2x80x2-methoxy thiophen-3-yl, or bycyclic systems such as indolyl, benzthiopheneyl, benzofuranyl, isoindolyl, isobenzothiophenyl, isobenzofuranyl
or alkyl carbamate of the formula xe2x80x94NHxe2x80x94CO-alkane, where in the alkane comprises (C1-6)alkyl, 4-methyl piperazinyl methyl,
or morpholino (C1-6)alkyl, piperazino (C1-6)alkyl,
or nitrilo (C1-6)acyl,
or an aryl, wherein the aryl comprises of any six membered aromatic carbocycle such as phenyl or a polycyclic aromatic hydrocarbon such as naphthyl, phenanthracenyl, indanyl
or a heterocycle wherein the heterocyle comprises of six membered aromatic heterocycles such as piridyl, diazinyl, pyrimidinyl, pyrrolidinyl, piperazinyl, or five membered aromatic heterocyles comprising pyrrolyl, pyrazole, imidazolyl , imidazolidinyl, imidazolenyl, oxazolyl, isoxazolyl, thiazolyl, thiazolidinyl, thiazolinyl, isothiazolyl, isothiazolidinyl, isothiazolinyl, furanyl, thiophenyl or bycyclic systems comprising indolyl, benzthiopheneyl,benzofuranyl, isoindolyl, isobenzothiophenyl, isobenzofuranyl; wherein the aryl or the heterocycle may each be unsubstituted or substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-8 alkylamino-C1-6 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl;
or C1-6 alkyl, aryl-C1-6 alkyl, mono- or polyfluorinated C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, cyclo(C3-6)alkyl, hydroxy (C1-6)alkyl, dihydroxy (C1-6)alkyl,
R4 comprises H, NH2, C1-6 alkyl, aryl-C1-6 alkyl, mono- or polyfluorinated C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl, cyclo(C3-6)alkyl,
or an aryl, wherein the aryl comprises of any six membered aromatic carbocycle such as phenyl or a polycyclic aromatic hydrocarbon such as naphthyl, phenanthracenyl, indanyl or a heterocycle wherein the heterocyle comprises of six membered aromatic heterocycles such as piridyl, diazinyl, pyrimidinyl, pyrrolidinyl, piperazinyl, or five membered aromatic heterocyles such as pyrrolyl, pyrazole, imidazolyl, imidazolidinyl, imidazolenyl, oxazolyl, isoxazolyl, thiazolyl, thiazolidinyl, thiazolinyl, isothiazolyl, isothiazolidinyl, isothiazolinyl, furanyl, thiophenyl or bycyclic systems such as indolyl, benzthiopheneyl, benzofuranyl, isoindolyl, isobenzothiophenyl, isobenzofuranyl; wherein aryl or heterocycle may each be unsubstituted or substituted with one or more of the following: C1-6 alkyl, C1-6 alkoxy, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-8 alkylamino-C1-8 alkyl, di(C1-6 alkyl)amino-C1-8 alkyl;
or a pharmaceutically acceptable salt thereof.
The preferred compounds are wherein R1 and R2 combine to form a methylenedioxy ring, R3 is H, and R4 is OH; or
Wherein R1 and R2 combine to form an ethylenedioxy ring, R3 is H, and R4 is OH; or
Wherein R1 and R2 combine to form an isopropylenedioxy ring, R3 is H, and R4 is OH; or
Wherein R1 and R2 are both CH3, R3 is H, and R4 is OH; or
Wherein R1 and R2 are both H, R3 is H, and R4 is OH.
Another object of the invention is to provide a method of inhibiting toposisomerase I activity in a mammal comprising administering to a mammal in need of inhibition of topoisomerase I activity an effective amount of a compound of the formula I or II.
Yet another object of the invention is to provide compositions for inhibiting topoisomerase I activity in a mammal in need of inhibition of topoisomerase I activity an effective amount of at least one compound of the formulas I and II.
These and other objects of the invention will be clear in light of the detailed description below.
The compounds disclosed in the present invention are useful as antitumor agents for the treatment or prevention of cancer, either alone or with a carrier. Cytotoxic agents are often employed as anticancer agents to control or eradicate tumors. Topo I poisons are useful cytotoxic agents, and two Topo I poisons related to camptothecin, Camptosar and Hycamtin (topotecan) are currently used clinically for the treatment of tumors. Indolocarbazoles are a different class of Topo I poison that represent useful agents for the treatment of tumors. In particular, Topo I-poisoning compounds disclosed in this invention were shown to be highly cytotoxic against human ovarian and prostate tumor cells.
Indolocarbazole analogues of this invention may be formulated as a solution of lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or in buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxycellulose, acacia, polyethylene glycol, mannitol, sodium chloride, or sodium acetate.
Alternatively, the compounds of the present invention may be encapsulated, tableted, or incorporated into an emulsion (oil-in-water or water-in-oil) syrup for oral administration. Pharmaceutically acceptable solids or liquid carriers, which are generally known in the pharmaceutical formulary arts, may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch (corn or potato), lactose, calcium sulfate dihydrate, terra alba, croscarmellose sodium, magnesium stearate or stearic acid, talc, pectin, acacia, agar, gelatin, maltodextrins and microcrystalline cellulose, or colloidal silicon dioxide. Liquid carriers include syrup, peanut oil, olive oil, corn oil, sesame oil, saline, and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 10 mg to about 1 g per dosage unit.
The dosage ranges for administration of indolocarbazole analogues disclosed in this invention are those to produce the desired affect. The dosage will generally vary with age, body weight, extent of the disease, and contraindications, if any. The dosage will also be determined by the existence of any adverse side effects that may accompany the compounds. It is always desirable, whenever possible, to keep adverse side effects to a minimum. One skilled in the art can easily determine the appropriate dosage, scheduling, and method of administration for the exact formulation of the composition being used in order to achieve the desired effective concentration in the individual patient. However, the dosage can vary from between about 1 mg/kg/day to about 500 mg/kg/day, and preferable from between about 1 mg/kg/day to about 50 mg/kg/day.
One skilled in the art will recognize that modifications may be made in the present invention without deviating from the spirit or scope of the invention. The invention is illustrated further by the following examples, which are not to be construed as limiting the invention in spirit or scope to the specific procedures or compositions described in them.

The required 5,6-indolodioxan and 5,6-indolodioxole precursors can be prepared starting from 4-methylcatechol, as illustrated in Scheme A for 5,6-ethylenedioxyindole (4). Protection of the ortho-dihydroxy function was achieved using 1,2-dibromoethane, dichloromethane, and acetone, respectively. Nitration using fuming nitric acid followed by indole formation using the Batcho-Leimgruber protocol12 afforded the desired indoles.
Construction of indolocarbazole analogues can be conducted as illustrated in Scheme B for Ia. N-Benzyloxymethyl-3,4-dibromomaleimide was prepared as previously described13. Reaction with an appropriate indole, which had been pre-treated using an organometallic, preferably but not limited to methylmagnesium halide or lithium hexamethyldisilazide, afforded the bromoindolomaleimide intermediates represented by 5. Glucosidation at the indole nitrogen was achieved with 2,3,4,6-tetra-O-benzyl-D-glucose under Mitsunobu conditions14 (3 equivalents each of the glucose, PPh3, and diisopropylazodicarboxylate (DIAD), followed by reversed-phase purification to afford 6. Introduction of the second indole unit was conducted under conditions similar to introduction of the first indole unit, providing the bis-indolylmaleimides represented by 7. Oxidative cyclization of the bis-indolylmaleimides was achieved using either palladium(II)trifluoroacetate in DMF, or via photochemical cyclization, providing indolocarbazoles represented by 8. Others14 reported oxidative cyclization using alternative reagents such as CuCl2 and PdCl2, but these failed to catalyze the reaction in our hands. Hydrogenolysis of the protective groups (palladium hydroxide, HOAc) afforded the 6-N-hydroxymethyl derivatives represented by 9, which were readily converted to the desired final products, represented by Ia, using ammonium acetate in methanol. All compounds provided spectral and analytical characteristics (1H NMR, 13C NMR, MS and elemental analysis) consistent with the targeted structures. 